~ steps get to our bacteria ~

we have been given a task to find our bacteria based on some information....

• part of DNA sequence
• part of protein sequence
• gene position
• and some other information

below are the steps to get approach to our lovely bacteria~~~ ♥

1. Go to BLAST website (click on the BLAST link)
   
2. Click the Nucleotide Blast which under the topic Basic Blast
   
3. Paste the partial DNA sequence on "Enter Query Sequence"
4. Choose Nucleotide collection (nr/nt) under "Choose Search Set -Database"
5. Click on BLAST when you are finish all the step above.
6. A list of bacteria or organisms will be listed out.
7. The 1st organism in the list is our LOVELY ORGANISM - Mycoplasma pneumoniae 's adhesion protein

LOCUS       EF614306                 825 bp    DNA     linear   BCT 18-MAR-2008
DEFINITION  Mycoplasma pneumoniae adhesin protein (P30) gene, complete cds.
ACCESSION   EF614306
VERSION     EF614306.1  GI:148729625
KEYWORDS    .
SOURCE      Mycoplasma pneumoniae
ORGANISM  Mycoplasma pneumoniae
Bacteria; Tenericutes; Mollicutes; Mycoplasmataceae; Mycoplasma.
REFERENCE   1  (bases 1 to 825)
FEATURES             Location/Qualifiers
source          1..825
   /organism="Mycoplasma pneumoniae"
   /mol_type="genomic DNA"
   /isolation_source="clinical sample"
   /db_xref="taxon:2104"
gene            1..825
   /gene="P30"
CDS             1..825
   /gene="P30"
   /codon_start=1
   /transl_table=4
   /product="adhesin protein"
   /protein_id="ABR09215.1"
   /db_xref="GI:148729626"
   /translation="MKLPPRRKLKLFLLAWMLVLFSALIVLATLILVQHNNTELTEVK
   SELSPLNVVLHAEEDTVQIQGKPITEQAWFIPTVAGCFGFSALAIILGLAIGLPIVKR
   KEKRLSEEKERQEQLAEQLQRISAQQEEQQALEQQAAAEAHAEAEVEPAPQPVPVPPQ
   PQVQINFGPRTGFPPQPGMAPRPGMPPHPGMAPRSGFPPQPGMAPRPGMPPHPGMAPR
   PGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMQPPRPGMPPQPGFPPKR"
ORIGIN
        1 atgaagttac cacctcgaag aaagcttaaa ctgtttttat tagcctgaat gctagtgctg
61 ttcagcgctt taatagtgct tgcaacctta attttggtac agcacaacaa taccgaactg
121 acagaagtta agagtgaatt gagtcccctt aacgttgttt tacacgcaga agaggataca
181 gtacaaattc agggcaagcc gattactgag caagcatggt ttattcctac agttgctggt
241 tgctttggtt ttagtgccct agccatcatc ttgggccttg ctataggact gccaattgtg
301 aagcgcaagg aaaaacgctt atcggaggaa aaggaacgcc aagaacagtt agcggaacag
361 ctacaacgca tttctgccca acaagaagag caacaagcgt tagaacaaca agcagctgct
421 gaagcccatg ctgaagcgga agttgaacca gcaccacaac cagtaccagt accacctcaa
481 ccccaagtcc aaattaactt cggtccccgt actggtttcc cacctcaacc cggtatggcg
541 cctcgtccag gtatgccgcc acaccccggt atggctccaa gatctggttt cccacctcaa
601 cccggtatgg cgcctcgtcc aggtatgccg ccacaccccg gtatggctcc aagacctggt
661 ttcccacctc aacccggtat ggcgcctcgt ccaggtatgc cgccacaccc cggtatggct
721 ccaagacctg gtttcccacc tcaacccggt atggcgcctc gtcccggaat gcaaccacca
781 cgtcctggca tgccacccca acccggtttt ccaccaaaac gctaa
//

finally~~ we get the protein~~~ and our bacteria ~~~ ♥

\(^O^)/

This diagram shows proteins in Mycoplasma pneumoniae and how they link with each other.
Adhesin protein, ATP synthase and other proteins are found at the cell membrane.
3D structure of the protein also can be seen in this diagram.

Gene Function of p30 in Mycoplasma Pneumonia

• Mycoplasma pneumoniae gene, p30 code for adhesin protein which associated with the attachment organelle of Mycoplasma pneumoniae, yet, its deep function is not clear.

• Needed for cytadherence (will attach Mycoplasma to epithelium) and it is virulence which will cause disease.

• P30 is a 29.7-kDa polypeptide, positively charged amino terminus and is followed by a hydrophobic domain of 23 residues which may act as a signal peptide.

• A second hydrophobic domain follows 40 residues after the first.
  

• However, P30 is predicted as highly hydrophilic.
  

• The C-terminal domain of P30 shows important sequence homology with the C terminus of the adhesin P1.
  

• P30 exhibits immunological cross-reactivity with fibrinogen, keratin, and myosin, which might used for autoimmune affect associated with Mycoplasma pneumoniae infections.
  

• Inability to cytadhere happened due to a complete loss of P30 (mutant II-3) or a 144-bp deletion near the 3′ hydroxyl end of the p30 gene (mutant II-7).
  

• Monoclonal antibody is used directly to against the extracellular domain of P30 which block its adherence, but this may reflect stearic interference with adjacent molecules on the Mycoplasma surface.

BioLoGy cHaRaCterIstiC of Mycoplasma pneumoniae

• Mycoplasma pneumoniae is completely lack of a bacterial cell wall

• Mycoplasma pneumoniae is osmotic fragility

• No colony shape

• Transferability through 450nm pore diameter membrane filters

• Low %mol G+C (guanine and cytosine) content DNA

Reservior & Transmission of Mycoplasma pneumoniae

There are 3 types of transmission of disease

a. contact

b. vehicles

c. vector

Contact transmission:
• is spread of an agent disease by direct contact, indirect contact or droplet transmission.

•Through kissing, touching, and sexual intercourse

• Droplet transmission occur in which microbes are spread in droplet nucleid that only travel in short distance.

Vehicle transmission:
• Transmission of disease agent by medium such as water, food, blood drug and intravenous fluids.

Vectors:
• Arthropods are the most important group of disease vectors

• They are animals that carry pathogens from one host to another.

The modes for Mycoplasma pneumoniae of transmission through:

A.respiratory droplet

B. airborne

A. respiratory droplet:

steps 1:

Mycoplasma pneumoniae is spread in the droplet nucleid that travel only a short distance.

steps 2:

these droplets area discharged into the air by coughing, sneezing, laughing or talking

steps 3:

they travel less than 1 meter from the reservoir to the host cell.

B. Airborne

- An enormous number of moisture droplets are expelled during sneezing.

- A considerable number of moisture droplets are expelled during coughing or talking.

- The infectious droplet of Mycoplasma pneumoniae has a diameter about 10µm.

- Each of the infectious droplets contains one or more microbial cell or virions.

- They can move in the speed of 100m/sec( more than 200mi/h) in sneeze.

- During coughing and talking, the range of speed will be 16 to 48m/sec.

- So, the number of bacteria in a sneeze will be around 10,000 to 100,000.

- The moisture droplets will evaporate and spread in the air very quickly as the size is small.

- The infectious droplets normally will cause upper respiratory infections such as influenza.

Reservoir is a continual source of infection. It may be human, animal or nonliving things.

1. Reservoir for Mycoplasma pneumoniae believed are human and mainly are children. People with signs and symtoms of a disease may transmit disease to others, they are called carrier. Mostly, children are living reservoir for Mycoplasma pneumoniae.
   
   
2. Another reservoir for Mycoplasma pneumoniae is wild animal such as deer, mice and rodents.

Reservoir: human

· Human body will be the reservoir for Mycoplasma pneumoniae.

· People can transmit the disease without exhibiting any signs and illness.

Reservoir : wild animals

· Disease that occur primary in wild and domestic animals and are able to transmit disease to human is called zoonoses.

· Mycoplasma pneumoniae present in most of wild animals such as deer and mice

sheep

pig

Epidemiology Information in Malaysia

Epidemiology: Study of factors of pathogens affect heath and illness of population, when, where and how they are transmitted in a population

Clinical Factors associated with Mycoplasma pneumoniae infection in 170 children admitted with Community Acquired Pneumonia

• Odd ratio (OR): Odd ratio is a measure of effect size which describes strength of association or non-independence between two binary data values. It is used as descriptive statics and plays an important role in logistic regression.

• Confident interval (CI): Interval estimate of a population parameter.

• p-value (p): Probability of observation due to chance.

The lower the p-value, the higher the statistical confident level.

• Serology positive = found antibody for Mycoplasma pneumoniae in serum of the particular people

• Serology negative = cannot found antibody for Mycoplasma pneumoniae in serum of the particular people

From the table, we can see that among 31 children among 40 children who are older than 3 years old were tested and were having antibody for Mycoplasma pneumoniae, 61 children among 130 children do not find antibody for Mycoplasma pneumoniae.

28 boys’ and 12 girls’ serum have found having the antibody for Mycoplasma pneumoniae. This means boys have a high chance to be infected by Mycoplasma pneumoniae. Among three races in Malaysia, Chinese has the most higher positive result for serology positive test which means have antibody to Mycoplasma pneumoniae in their blood.

Statistic of Detection of Mycoplasma pneumoniae in adult patients and pediatric patient

• Pediatric: Medical care of children
  

• This statistic shows that Infection of Mycoplasma pneumoniae appear more frequently among children.
• Among 20 adult patient, there is only a adult infected by Mycoplasma pneumoniae. But, there are 15 children among 20 children affected by Mycoplasma pneumoniae.
  

World distribution of Pneumonia disease

Around the world, Africa is the largest portion that its resident having Pneumonia.

References:
Chan PW, Lum LC, Ngeow YF, Yasim MY., Jun 2001, Mycoplasma pneumoniae infection in Malaysian children admitted with community acquired pneumonia, PubMed

http://www.ncbi.nlm.nih.gov/pubmed/11556595

http://72.14.235.132/search?q=cache:23ihV7o1zKAJ:www.tm.mahidol.ac.th/seameo/2001_32_2/28-2711.pdf+mycoplasma+pneumoniae+epidemiology+malaysia&cd=4&hl=en&ct=clnk&gl=my&client=firefox-a

Diagnosis of Mycoplasma pneumoniae

Diagnosis - Identification of the characteristic of something, either by process of elimination or other analytical methods. Diagnosis is used widely to determine causes of symptomes, mitigations for problems, or solutions for particular issue.

Below are methods that used to diagnose Mycoplasma pneumoniae which infect human body:

1. Stethoscope

• Method use to listen and check whether the patient produce abnormal sounds due the Mycoplasma pneumoniae infections.

• Not good enough because sometime it may indicate there is no infection even when Mycoplasma pneumoniae actually present in the lung of the patients.

2. X-rays

• Patients have to undergo X-rays examination to check the lungs to confirm a diagnosis of Mycoplasma pneumoniae.

• For further confirm Mycoplasma pneumoniae, doctors may also take blood samples and check for Mycoplasma antibodies.

3. IgM serology test

• Cold-hemagglutinins test on the patient also can be done to check for Mycoplasma pneumoniae infection. Cold-hemagglutinins test is a type of serology test for Mycoplasma pneumoniae infection . The patients specimens are collected using a throat swabs by rubbing with a sterile swab over the posterior portion of pharynx after coughing. The collected samples are test with Mycoplasma pneumoniae-IgM antibody. The patients samples with Mycoplasma pneumoniae infection will causes the IgM antibodies to appear. Thus, this indicate a positive result for IgM antibodies and there are Mycoplasma pneumoniae infection in the lungs of the patients.

4. Blood agar

• Besides that, blood agar are used to culture the sample collected from patients. Mycoplasma pneumoniae do not grow well on blood agar. Thus, it help to diagnose the Mycoplasma pneumoniae infection by observe the growth on the agar.

5. Polymerase chain reaction (PCR)

• Polymerase chain reaction (PCR) is also used to diagnose Mycoplasma pneumoniae infection. It is used to amplify a single or few copies of a piece of DNA across several orders of magnitude and generate millions or more copies of a particular DNA sequence. PCR permits identification of slow-growing microorganisms such as Mycoplasma pneumoniae.The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and also the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

Pathogenesis caused by Mycoplasma pneumoniae

• Mycoplasma pneumoniae contains an adhesive protein that attaches itself to specific receptors that are located at the bases of cilia on the epithelial cells lining of the human respiratory tracts.

• The bacterium will extract nutrient from the mucosa of host organism to allow them grow and reproduce quickly.

• Mycoplasma pneumoniae undergoes asexual reproduction by binary fission. A single cell will grow until a maximum size and then start to undergo cell division which separate the original cell into two. The two cells then grow and reproduce again to produce a bacterium colony.

• The attachment of the bacteria causes the cilia of human respiratory tract to stop beating.

• Besides that, the bacteria colonization also kills the epithelial cells of the respiratory tract.

• This allows other bacteria to colonize the respiratory tract and causes mucus to accumulate and irritates the respiratory tract.

• There are a few early symptoms of Mycoplasma pneumoniae infections – fever, sore throat, and headache.
  

• The affected person will also have unproductive cough in attempt to clear the lungs.

• Affected person may get the infection for several weeks but it is usually not severe enough to cause death.
  

• Mycoplasma pneumoniae affects human respiratory tract through respiratory droplet transmission . Respiratory droplet transmission is transferred when an infected person sneezes or coughs to the air. They produce moist and warm droplets that may enter to other people body through the nose, mouth and eye surfaces.

• The infection caused by this bacterium is called atypical pnemonia which describe a form a of pneumonia not caused by one of the more traditional pathogens . This bacterium infection is lack of sputum production and wealth of extra-pulmonary symptoms.

PrEveNtioN and TreATmENt of pnEUmonIA cAUsed by Mycoplasma pneumoniae

Prevention:
~Practice good hygiene.

~Get an influenza shot each fall.

~Get a pneumonococcal vaccine. People who stand to benefit most from vaccination are those over the age 65; anyone with chronic health problems (such as diabetes, kidney disease, heart disease, etc.); anyone who has had their spleen removed; anyone living in a nursing home or chronic care facility; caregivers of the chronically ill (healthcare workers or family caregivers); children with chronic respiratory diseases (such as asthma), and anyone who has had pneumonia in the past (due to increased risk of reinfection). The pneumonococcal vaccine is 90 percent effective against the bacteria and protects against infection for five to 10 years.

~Practice good preventive measures by eating a proper diet, getting regular exercise and plenty of sleep.

~Do not smoke

Treatment:
~often treated with Antibiotics such as erythromycin, clarithromycin or azithromycin are effective treatment.

~However, because mycoplasma infection usually resolves on its own, antibiotic treatment of persons with mild symptoms is usually not necessary.

~ Erythromycin250mg PO four times a day or doxycycline 100mg PO twice-daily are traditional and inexpensive choices.

~Newer, more expensive drugs with excellent in vitro MIC's include: azithromycin 250mg PO once-daily, clarithromycin250mg PO q12h, levofloxacin 250mg PO once-daily, moxifloxacin 400mg PO once-daily.

Genome Information for Mycoplasma pneumoniae

Kingdom: Bacteria
Division: Firmicutes
Class: Mollicutes
Order: Mycoplasmatales
Family: Mycoplasmataceae
Genus: Mycoplasma

• Mycoplasma pneumoniae was linked to respiratory infection by Roux and Nocard in 1989. Mycoplasma pneumoniae is the bacteria spread through respiration system and usually causes infection.

• It was found when Roux and Nocard isolated the organisms from bovine pleuropneumonia specimens (another respiratory system infectious caused by other bacteria).

• The infection causes by Mycoplasma pneumoniae such as tracheobranchitis and primary atypical pneumonia.

• Mycoplasma pneumoniae is characterized by small genome and their lack of cell walls.

• Mycoplasma pneumoniae has a genome of 800kb which has been mapped by researchers in germany in Richard Hermann's laboratory.

Mycoplasma pneumoniae chromosome mapped using EcoRI.

• Based on the research that done by Richard Hermann in the Genome Project, it was proved that the genome size of Mycoplasma pneumoniae is 816394bp long.

• The length of the Mycoplasma pneumoniae's coding regions is 724174bp and it corresponds to 88.7% of the entire genome.

Mycoplasma pneumonia genome

New Diagnostic Strategy

OK~Colonization, host and bacterial, factors contribute to progression of infection are important to optimize diagnosis and treatment, and to prevent transmission of Mycoplasma pneumoniae.

• Survey need to be done from time to time in the countries that normally Mycoplasma pneumoniae diseases occurred.
  

• Differentiating between asymptomatic colonization and infection using quantitative, real-time PCRS. Via PCR, small samples of DNA can be quickly amplified to increase the number of samples so that there are enough for analysis. By using the PCR, diagnosis test can use to detect the presence of infections agents in situations in which they would otherwise be undetectable.

• DNA fingerprinting to track an infection disease. This procedure enables the identification of a particular DNA sequence. Determination and identification particular DNA by southern blotting is commonly used; it is used to detect whether bacteria’s DNA are present in the white blood cell DNA of a patient by comparing the DNA content of normal cell with infected white blood cell.

• Medical examination on host’s white blood cell whether have any similar symptom with Mycoplasma pneomoniae cytadherence.
  

• By using and introducing new Mycoplasma that having a new coding gene on adhesin protein into the lungs. Pathogenic Mycoplasma will rearrange DNA by using multiple copies of adhesion gene sequences to replace their natural disadvantage which is having a small genome. Gene reshuffles also a high rate reinjection of Mycoplasma pneumoniae into patients. It is able to activate anti-self T cells or polyclonal B cells. So, from our blood sample, white blood cell gene and normal white blood cell gene are compared in order to check whether there are any differences between both genetic coding. If the result is positive it means Mycoplasma pneumoniae is present.

• A new device which is able to scan and print the image of the patient lungs can be created based on fluorescence principle. Fluorescence probe that react with Mycoplasma pneumoniae will be bring into respiratory tract, then image of the patient’s lungs will be captured and printed. Picture that shows fluorescence proved that Mycoplasma pneumoniae presence.

• An advanced technology device can be made. An advanced bronchoscope is modified to add a gas collection tube and remote sample collection hook on it. When doctor using this tool, it can capture situation in the respiratory tract and can collect the gas surround the infected area; infection is found, doctor can use the remote hook collect the infectious sample.

• The little tube will have some little holes and are covered by soft materials. It will contain a low pressure gas, when the tube insert into the respiratory tract, high pressure will enter the tube thru the hole. If the gas collect turn colorless 2,3,5-triphenyl tetrazolium chloride into pink color. We can do further checking on it. Bacteria have to grow and undergo some test. If it fermented glucose, turn glucose nutrient broth methyl-red indicator from red to yellow and give a positive serology IgM result, then we can predict Mycoplasma pneumoniae is found.